Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
Primer design results for 'tail' primers (this is just 2 sequences):
FORWARD PRIMER TACTTCCAATCCATGAAGAAAATTTCCGTTCTG REVERSE PRIMER TATCCACCTTTACTGTTAAGAGTACGCCAGAAC Underlined: 18 bp from CDS Highlighted: start and stop codons
Primer Sequence for 1-deoxy-D-xylulose 5- phosphate reductoisomerase (Wolbachia endosymiont strain TRS of Brugia Malayi)
NCBI Gene # or RefSeq#: 3188958
Protein ID (NP or XP #) or Wolbachia#: Q5GTA4
MHS Rank: 106 GCS Rank: 476 on WOLTR spreadsheet
Organism: Wolbachia
Etiologic Risk Group (see link below): none listed
Background/Disease Information: Wolbachia infects isopods, mites, spiders, and nematodes. The filarial worms Wolbachia infects cause diseases such as heartworm in dogs and River Blindness and Elephantitis in humans
Essentiality of this protein: http://tdrtargets.org/targets/view?gene_id=233342
essential in 3 studies done on E.coli
Complex of enzymes: No
EC#: 1.1.1.267
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.267&Suchword=&organism%5B%5D=Escherichia+coli&show_tm=0
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
http://tdrtargets.org/targets/view?gene_id=233342
No assay found for Wolbachia.
Spectrophotometric assay done in Pseudomonas aeruginosa.
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/search?interface=All&term=1-Deoxy-D-xylulose-5-phosphate&lang=en®ion=US&focus=product&N=0+220003048+219853269+219853286&mode=match%20partialmax
1-Deoxy-D-xylulose-5-phosphate sodium salt
Product #: 13368
1 mg costs $66.30
-- link to paper that contains assay information not found
Structure Available (PDB or Homology model): http://www.rcsb.org/pdb/explore/explore.do?structureId=1r0k
-- PDB # or closest PDB entry if using homology model: 1R0K (organism: Zymomonas mobilis)
-- Query Coverage (if not direct match): 98%
-- Max Ident (if not direct match): 43%
Druggable Target (see Databases for this):
http://tdrtargets.org/targets/view?gene_id=233342
Known druggable targets in E.coli and synechocystis
Current Inhibitors:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.267&Suchword=&organism%5B%5D=Escherichia+coli&show_tm=0
(in E.coli)
fosmidomycin .0035mM
(2R,3R)-2,3,4-trihydroxybutyl dihydrogen phosphate .310 mM IC50
Expression Information (has it been expressed in bacterial cells):
none found
Purification Method:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.267&Suchword=&organism%5B%5D=Escherichia+coli&show_tm=0
Image of protein (PyMol or etc):
1ROK, PDB
Amino Acid Sequence:
http://www.uniprot.org/uniprot/Q5GTA4
>sp|Q5GTA4|DXR_WOLTR 1-deoxy-D-xylulose 5-phosphate reductoisomerase OS=Wolbachia sp. subsp. Brugia malayi (strain TRS) GN=dxr PE=3 SV=1 MKKISVLGSTGSIGKKTVDLLLKRKEEYQVEALSTCSNFALLACQAKLLNARYVAISNKR FYKDLKESLLGTGIKVEVGTEGLMNVASLPVDLSVVAVVGIAGLEPVMHVIESGTKVIAL ANKESIVCGGKLLLKKTEEKNVQIVPIDSEHNAIFQVLQNGNKCVEKIILTASGGSFLNY SLEQLRNVTVGQALSHPTWNMGKKISVDSATMMNKALEIIEAHNLFNISPNRIEAIVHPE SIIHGIVIYKDGFNFAVLAEADMAIPISYALSWPERSALSYKLDLTKQKLTFQEPDHKRF PALKLSMEVLNSSSPHTNSIVLNAANEVAVDKFLKSRIDFLEIIKVVKLTVENFDSYTDI NSLSDIINIDLESRAIAKEIIKNKVLAYS
<span style="background-color: #f5f5f5; text-align: left;"> 1**__0__** 2**__0__** 3**__0__** 4**__0__** 5**__0__** 6**__0__** MKKISVLGST GSIGKKTVDL LLKRKEEYQV EALSTCSNFA LLACQAKLLN ARYVAISNKR 7**__0__** 8**__0__** 9**__0__** 10**__0__** 11**__0__** 12**__0__** FYKDLKESLL GTGIKVEVGT EGLMNVASLP VDLSVVAVVG IAGLEPVMHV IESGTKVIAL 13**__0__** 14**__0__** 15**__0__** 16**__0__** 17**__0__** 18**__0__** ANKESIVCGG KLLLKKTEEK NVQIVPIDSE HNAIFQVLQN GNKCVEKIIL TASGGSFLNY 19**__0__** 20**__0__** 21**__0__** 22**__0__** 23**__0__** 24**__0__** SLEQLRNVTV GQALSHPTWN MGKKISVDSA TMMNKALEII EAHNLFNISP NRIEAIVHPE 25**__0__** 26**__0__** 27**__0__** 28**__0__** 29**__0__** 30**__0__** SIIHGIVIYK DGFNFAVLAE ADMAIPISYA LSWPERSALS YKLDLTKQKL TFQEPDHKRF 31**__0__** 32**__0__** 33**__0__** 34**__0__** 35**__0__** 36**__0__** PALKLSMEVL NSSSPHTNSI VLNAANEVAV DKFLKSRIDF LEIIKVVKLT VENFDSYTDI 37**__0__** 38**__0__**NSLSDIINID LESRAIAKEI IKNKVLAYS</span>TMPREDput TmPred result image here .............
http://www.ch.embnet.org/software/TMPRED_form.html
http://www.ch.embnet.org/cgi-bin/TMPRED_form_parser
length of your protein in Amino Acids: 389
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite
http://web.expasy.org/cgi-bin/protparam/protparam1?Q5GTA4@1-389@
Molar Extinction coefficient of your protein at 280 nm wavelength:
CDS Gene Sequence:
1167 bp
http://www.ncbi.nlm.nih.gov/gene?term=Wbm0179
GC% Content for gene: 35.45%
http://bioinformatics.org/sms2/dna_stats.html
CDS Gene Sequence (codon optimized) - after having done Primer Design:
https://docs.google.com/file/d/0B4O2KqKh2q_-NjNoY1pxcUw5OXc/edit
ATGAAGAAAATTTCCGTTCTGGGTTCTACCGGCTCTATCGGTAAAAAGACCGTAGATCTGCTGCTGAAACGCAAAGAAGAATACCAGGTTGAAGCGCTGTCTACCTGCTCTAACTTCGCGCTGCTCGCGTGTCAGGCGAAACTGCTCAATGCCCGCTATGTTGCGATCTCTAACAAACGCTTTTACAAAGACCTGAAAGAGTCTCTGCTGGGTACCGGCATTAAAGTTGAAGTTGGTACCGAAGGTCTGATGAACGTAGCGTCTCTCCCGGTTGACCTGTCTGTTGTCGCAGTTGTTGGTATCGCGGGTCTGGAACCAGTTATGCATGTCATCGAATCTGGTACGAAAGTTATCGCGCTCGCGAACAAAGAATCTATCGTTTGCGGTGGTAAACTCCTCCTCAAGAAAACTGAAGAGAAAAACGTCCAGATCGTGCCGATCGACAGCGAGCACAACGCGATCTTCCAGGTACTGCAAAACGGCAACAAATGTGTCGAAAAAATCATCCTGACCGCGTCTGGTGGTTCCTTCCTGAATTACTCTCTCGAACAGCTGCGTAATGTTACCGTAGGTCAGGCCCTGTCTCACCCGACGTGGAACATGGGCAAAAAGATCTCCGTTGACTCTGCGACCATGATGAATAAGGCGCTCGAAATTATCGAGGCACACAACCTGTTCAACATCTCTCCGAACCGTATCGAAGCGATCGTTCACCCGGAGTCTATCATCCACGGTATCGTTATCTACAAGGACGGTTTCAATTTCGCCGTGCTGGCGGAAGCGGACATGGCGATCCCGATTTCTTATGCACTGTCTTGGCCGGAGCGTTCCGCGCTGAGCTACAAACTGGACCTGACCAAACAGAAACTGACTTTCCAAGAACCGGACCATAAACGCTTCCCGGCACTGAAACTGTCTATGGAAGTTCTCAACTCTTCTTCTCCGCACACCAACAGCATCGTTCTGAACGCTGCGAACGAAGTTGCAGTGGACAAATTTCTGAAATCTCGCATCGATTTCCTGGAGATTATCAAAGTTGTTAAACTCACCGTTGAAAACTTCGACTCTTATACCGACATCAACTCCCTCTCTGACATCATTAACATTGACCTCGAAAGCCGTGCGATTGCAAAAGAGATCATCAAAAACAAAGTTCTGGCGTACTCTTAA
GC% Content for gene (codon optimized):
http://bioinformatics.org/sms2/dna_stats.html
48.46%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
Primer design results for 'tail' primers (this is just 2 sequences):
FORWARD PRIMER TACTTCCAATCCATGAAGAAAATTTCCGTTCTG
REVERSE PRIMER TATCCACCTTTACTGTTAAGAGTACGCCAGAAC
Underlined: 18 bp from CDS
Highlighted: start and stop codons
Primer Sequence for 1-deoxy-D-xylulose 5- phosphate reductoisomerase (Wolbachia endosymiont strain TRS of Brugia Malayi)
https://docs.google.com/file/d/0B4O2KqKh2q_-NjNoY1pxcUw5OXc/edit
see oligonucleotide sequences